Methods for the reduction of stutter in microsatellite amplification

ABSTRACT

The invention provides a method for reducing stutter in the amplification of a microsatellite comprising the steps of providing a sample comprising a microsatellite having a G+C content of greater than 50%; contacting the sample with at least one enzyme having nucleic acid polymerase activity; and incubating the sample with the enzyme for a sufficient amount of time and under conditions sufficient to amplify the microsatellite; wherein the incubation is performed in the presence of an amount of sorbitol effective to reduce stutter relative to the amount of stutter observed in the absence of sorbitol. The invention also provides compositions containing sorbitol, kits for amplifying microsatellites having a G+C content of greater than 50%, and methods of using all of the foregoing.

FIELD OF THE INVENTION

[0001] The invention relates to methods, compositions and kits forreducing stutter in polymerase chain reaction amplification ofmicrosatellites. In certain embodiments, the invention relates to theuse of sorbitol in polymerase chain reactions in an amount effective toreduce stutter in the amplification of mononucleotide, dinucleotide,trinucleotide, tetranucleotide and pentanucleotide microsatellites.

BACKGROUND OF THE RELATED ART

[0002] Microsatellites, or short tandem repeats (STRs), consist oftandemly repeated DNA sequence motifs of 1 to 6 nucleotides in length.They are widely dispersed and abundant in the eukaryotic genome, and areoften highly polymorphic due to variation in the number of repeat units.This polymorphism renders microsatellites attractive DNA markers forgenetic mapping, medical diagnostics and forensic investigation. Thecombination of PCR and gel or capillary electrophoresis under denaturingconditions has greatly improved the genotyping of microsatellite DNAsequences. However, PCR artifacts exhibited by non-proofreading enzymesand referred to as stutter and the terminal transferase side-reactioncan complicate analysis of closely spaced microsatellite alleles.

[0003] Stutter signals differ from the PCR product representing thegenomic allele by multiples of repeat unit size. For dinucleotide repeatloci, the prevalent stutter signal is generally two bases shorter thanthe genomic allele signal, with additional side-products that are 4 and6 bases shorter. The multiple signal pattern observed for each alleleespecially complicates interpretation when two alleles from anindividual are close in size (e.g., medical and genetic mappingapplications) or when DNA samples contain mixtures from two or moreindividuals (e.g., forensic applications). Such confusion is maximal formononucleotide microsatellite genotyping, when both genomic and stutterfragments experience one-nucleotide spacing.

[0004] There is a need in the art to develop PCR reaction conditionsthat minimize or eliminate stutter so that genetic analysis may be moreaccurate and reliable. This invention is directed to these, as well asother, important ends.

SUMMARY

[0005] In accordance with some embodiments of the methods of theinvention, methods for reducing stutter in the amplification of amicrosatellite are provided comprising the steps of:

[0006] (a) providing a sample comprising a microsatellite of interest,in which the microsatellite has a G+C content of greater than 50%;

[0007] (b) contacting the sample with at least one enzyme having nucleicacid polymerase activity; and

[0008] (c) incubating the sample with the enzyme for a time and underconditions sufficient to amplify the microsatellite;

[0009] wherein said incubation is performed in the presence of an amountof sorbitol effective to reduce stutter relative to the amount ofstutter observed in the absence of sorbitol.

[0010] The invention also provides methods for reducing stutter in theamplification of a mononucleotide microsatellite comprising the stepsof:

[0011] (a) providing a sample comprising a microsatellite of interest,in which the microsatellite has a G+C content of greater than 50%;

[0012] (b) contacting the sample with at least one enzyme having nucleicacid polymerase activity; and

[0013] (c) incubating the sample with the enzyme for a time and underconditions sufficient to amplify the microsatellite;

[0014] wherein the incubation is performed in the presence of an amountof sorbitol, wherein the sorbitol is effective to reduce stutterrelative to the amount of stutter observed in the absence of sorbitol.

[0015] The invention also provides methods for reducing stutter in theamplification of a dinucleotide microsatellite comprising the steps of:

[0016] (a) providing a sample comprising a microsatellite of interest,in which the microsatellite has a G+C content of greater than 50%;

[0017] (b) contacting the sample with at least one enzyme having nucleicacid polymerase activity; and

[0018] (c) incubating the sample with the enzyme for a time and underconditions sufficient to amplify the microsatellite; wherein theincubation is performed in the presence of an amount of sorbitoleffective to reduce stutter relative to the amount of stutter observedin the absence of sorbitol.

[0019] The invention further provides methods for reducing stutter inthe amplification of a trinucleotide microsatellite comprising the stepsof:

[0020] (a) providing a sample comprising a microsatellite of interest,in which the microsatellite has a G+C content of greater than 50%;

[0021] (b) contacting the sample with at least one enzyme having nucleicacid polymerase activity; and

[0022] (c) incubating the sample with the enzyme for a time and underconditions sufficient to amplify the microsatellite; wherein theincubation is performed in the presence of an amount of sorbitoleffective to reduce stutter relative to the amount of stutter observedin the absence of sorbitol.

[0023] The invention further provides methods for reducing stutter inthe amplification of a tetranucleotide microsatellite comprising thesteps of:

[0024] (a) providing a sample comprising a microsatellite of interest,in which the microsatellite has a G+C content of greater than 50%;

[0025] (b) contacting the sample with at least one enzyme having nucleicacid polymerase activity; and

[0026] (c) incubating the sample with the enzyme for a time and underconditions sufficient to amplify the microsatellite; wherein theincubation is performed in the presence of an amount of sorbitoleffective to reduce stutter relative to the amount of stutter observedin the absence of sorbitol.

[0027] The invention further provides methods for reducing stutter inthe amplification of a pentanucleotide microsatellite comprising thesteps of:

[0028] (a) providing a sample comprising a microsatellite of interest,in which the microsatellite has a G+C content of greater than 50%;

[0029] (b) contacting the sample with at least one enzyme having nucleicacid polymerase activity; and

[0030] (c) incubating the sample with the enzyme for a time and underconditions sufficient to amplify the microsatellite; wherein theincubation is performed in the presence of an amount of sorbitoleffective to reduce stutter relative to the amount of stutter observedin the absence of sorbitol.

[0031] In further embodiments of the methods of the invention, methodsare provided comprising the steps of:

[0032] (a) providing a sample comprising a nucleic acid that containsone or more microsatellites selected from the group consisting ofmononucleotide microsatellites, dinucleotide microsatellites,trinucleotide microsatellites, tetranucleotide microsatellites andpentanucleotide microsatellites; and

[0033] (b) amplifying at least one nucleobase sequence of said nucleicacid, said nucleobase sequence comprising at least one of saidmicrosatellites; said amplified microsatellite having a G+C content ofgreater than 50%; wherein said amplification is performed in thepresence of sorbitol.

[0034] Also provided in accordance with the present invention aremethods for performing polymerase chain reaction amplification of amicrosatellite selected from the group consisting of mononucleotidemicrosatellites, dinucleotide microsatellites, trinucleotidemicrosatellites, tetranucleotide microsatellites and pentanucleotidemicrosatellites, said microsatellite having a G+C content of greaterthan 50%; said method comprising the step of contacting saidmicrosatellite with a polymerase in the presence of an amount ofsorbitol effective to reduce the amount of stutter arising from saidamplification relative to the amount of such stutter observed in theabsence of sorbitol.

[0035] Also provided by the present invention are methods of detectingcancer or a pre-cancerous condition and genetic disorders, in a subjectcomprising amplifying a region of DNA from a subject, wherein saidregion comprises a microsatellite selected from the group consisting ofa mononucleotide repeat, a dinucleotide repeat, a trinucleotide repeat,a tetranucleotide repeat and a pentanucleotide repeat, wherein saidamplification comprises the steps of:

[0036] (a) providing a sample comprising a nucleic acid that contains anucleic acid having a microsatellite instability,

[0037] (b) amplifying at least one nucleobase sequence of the nucleicacid, in which the nucleobase sequence comprises at least one of themicrosatellites; and

[0038] (c) detecting alterations of the microsatellite as compared tocorresponding microsatellites amplified from control tissue; theamplified microsatellite having a G+C content of greater than 50%;wherein the amplification is performed in the presence of a sufficientamount of sorbitol effective to reduce stutter from that observed in theabsence of sorbitol.

[0039] In some embodiments, the cancer or cancerous condition is chroniclymphocytic leukemia. In further embodiments, the microsatelliteamplification comprises at least one genetic locus, for example,CAG/CTG, CCG/CGG, and CGA/TCG.

[0040] In some embodiments, the genetic disorder is Huntington's diseaseor a spinocerebellar ataxia. In further embodiments, the microsatelliteamplification comprises at least one genetic locus, for example,CAG/CTG.

[0041] In other embodiments, the disorder is a psychiatric disorder. Infurther embodiments, the microsatellite amplification comprises at leastone genetic locus, for example, CCCCT/AGGGG.

[0042] In some embodiments, the present invention also provides methodsof genetic mapping comprising amplifying a plurality of regions of DNAfrom a sample containing DNA from a subject, wherein the regionscomprise at least one microsatellite selected from the group consistingof a mononucleotide repeat, a dinucleotide repeat, a trinucleotiderepeat, a tetranucleotide repeat and a pentanucleotide repeat, whereinthe amplification comprises the steps of:

[0043] (a) contacting said DNA with a enzyme at least one enzyme havingnucleic acid polymerase activity; and

[0044] (b) incubating said sample with the enzyme for a time and underconditions sufficient to amplify the regions; and

[0045] (c) separating amplified regions, forming a microsatellitepattern; wherein the incubation is performed in the presence of anamount of sorbitol effective to reduce stutter relative to the amount ofstutter observed in the absence of sorbitol.

[0046] In further embodiments, the present invention also providesmethods of personal genetic identification comprising amplifying aplurality of regions of DNA from a sample containing DNA from a subject,wherein said regions comprise at least one microsatellite selected fromthe group consisting of a mononucleotide repeat, a dinucleotide repeat,a trinucleotide repeat, a tetranucleotide repeat and a pentanucleotiderepeat; wherein the microsatellite has a G+C content of greater than50%; wherein the amplification comprises the steps of:

[0047] (a) contacting said DNA with a enzyme at least one enzyme havingnucleic acid polymerase activity; and

[0048] (b) incubating the sample with the enzyme for a time and underconditions sufficient to amplify the regions;

[0049] (c) separating amplified regions, forming a microsatellitepattern; and

[0050] (d) comparing the microsatellite pattern with a correspondingmicrosatellite pattern derived from the a DNA sample from a secondsource; wherein the incubation is performed in the presence of an amountof sorbitol effective to reduce stutter relative to the amount ofstutter observed in the absence of sorbitol.

[0051] In some embodiments, the subject is a forensic sample and a saidsecond source comprises at least one selected from the group consistingof the presumed matching source, a family member of the presumedmatching source, and a database of sources.

[0052] In some embodiments of the methods of the invention, where themicrosatellite is a dinucleotide microsatellite, the microsatellitecomprises a dinucleotide repeat is CG/CG. In further embodiments wherethe microsatellite is a trinucleotide microsatellite, the microsatellitecomprises a trinucleotide repeat selected from the group consisting ofCAG/CTG, CCG/CGG, and CGA/TCG. In further embodiments where themicrosatellite is a tetranucleotide microsatellite, the microsatellitecomprises a tetranucleotide repeat consisting of TGCC/GGCA. In furtherembodiments where the microsatellite is a pentanucleotidemicrosatellite, the microsatellite comprises a pentanucleotide repeatconsisting of CCCCT/AGGGG.

[0053] In some embodiments of the method of the invention, themicrosatellite has a G+C content of greater than 50%. In furtherembodiments, the microsatellite has a G+C content of greater than 66%.In further embodiments, the microsatellite has a G+C content of 75% ormore. In further embodiments, the microsatellite has a G+C content of100%.

[0054] In some embodiments of the invention, the amount of stutter isreduced to 90% or less than the amount of stutter obtained in theabsence of sorbitol. In other embodiments the amount of stutter isreduced to 80% or less. In other embodiments, the amount of stutter isreduced to 70% or less. In other embodiments, the amount of stutter isreduced to 60% or less. In other embodiments, the amount of stutter isreduced to 50% or less. In other embodiments, the amount of stutter isreduced to 40% or less. In other embodiments, the amount of stutter isreduced to 30% or less.

[0055] In some embodiments of the methods of the invention, theamplification comprises contacting said nucleobase sequence with anenzyme having a polymerase activity. For example, the enzyme havingpolymerase activity may be selected from the group consisting of a DNApolymerase from Thermus aquaticus, Thermus thermophilus, other Thermusspecies, Bacillus species, Thermococcus species, Thermotoga species, andPyrococcus species. For example, suitable polymerases include AmpliTaqGold® DNA polymerase; AmpliTaq® DNA Polymerase; AmpliTaq® DNAPolymerase, Stoffel fragment; rTth DNA Polymerase; rTth DNA PolymeraseXL; Bst DNA polymerase large fragment from Bacillus stearothermophilus;Vent and Vent Exo- from Thermococcus litoralis; Tma from Thermotogamaritima; Deep Vent and Deep Vent Exo- and Pfi from Pyrococcus; andmutants, variants and derivatives thereof.

[0056] Also provided in certain embodiments of the invention arecompositions comprising:

[0057] (a) a nucleic acid sequence comprising a microsatellite, in whichthe microsatellite has a G+C content of greater than 50%, themicrosatellite being selected from the group consisting ofmononucleotide microsatellites, dinucleotide microsatellites,trinucleotide microsatellites, tetranucleotide microsatellites andpentanucleotide microsatellites;

[0058] (b) at least two primers, each of said primers having a sequencethat is substantially complementary to a portion of the nucleic acidsequence that is adjacent to the microsatellite;

[0059] (c) at least one enzyme having nucleic acid polymerase activity;and (d) sorbitol.

[0060] In some embodiments of the methods and compositions of theinvention, sorbitol is present in an amount of from 1.5 to 3.5 M. Inother embodiments, sorbitol is present in an amount of 2.0 to 3.0 M. Inother embodiments, sorbitol is present in an amount of 2.0 M.

[0061] In some embodiments of the invention, at least 0.5 mM each ofdNTPs are used. In other embodiments, at least 1 mM dNTPs are used.

[0062] In some embodiments, the present invention also provides kits foramplification of a target nucleic acid sequence, the target nucleic acidsequence comprising a microsatellite having a G+C content of greaterthan 50%, selected from the group consisting of mononucleotidemicrosatellites, dinucleotide microsatellites, trinucleotidemicrosatellites, tetranucleotide microsatellites, and pentanucleotidemicrosatellites comprising, in separate containers: a polymerase, aplurality of deoxynucleotide triphosphates; and sorbitol. In someembodiments of the compositions and kits of the invention, thepolymerase is selected from the group consisting of a DNA polymerasefrom Thermus aquaticus, Thermus thermophilus, other Thermus species,Bacillus species, Thermococcus species, Thermotoga species, andPyrococcus species. For example, suitable polymerases include, but arenot limited to, AmpliTaq Gold® DNA polymerase; AmpliTaq® DNA Polymerase;AmpliTaq® DNA Polymerase, Stoffel fragment; rTth DNA Polymerase; rTthDNA Polymerase XL; Bst DNA polymerase large fragment from Bacillusstearothermophilus; Vent and Vent Exo- from Thermococcus litoralis; Tmafrom Thermotoga maritima; Deep Vent and Deep Vent Exo- and Pfu fromPyrococcus; and mutants, variants and derivatives thereof.

[0063] In some further embodiments of the invention, methods areprovided in which a sample containing nucleic acid that is suspected ofcontaining one or more microsatellites having a G+C content of greaterthan 50% is contacted with an enzyme that polymerizes nucleotides in thepresence of an effective amount of sorbitol to reduce observed stutterrelative to the amount of stutter observed in the absence of sorbitol,and amplifying at least one nucleobase sequence containing at least onemicrosatellite of the nucleic acid contained in the sample. Suchmicrosatellites may include mononucleotide, dinucleotide, trinucleotide,tetranucleotide microsatellites and/or pentanucleotide microsatellites.

BRIEF DESCRIPTION OF THE FIGURE

[0064]FIG. 1 shows GeneScan traces from PCR amplifications of atetranucleotide microsatellite ((CTTT/AAAG)_(n)) for control conditions(upper panel) and with added sorbitol (lower panel).

DETAILED DESCRIPTION

[0065] Most of the words used in this specification have the meaningthat would be attributed to those words by one skilled in the art. Wordsspecifically defined in the Specification have the meaning provided inthe context of the present invention as a whole, and as are typicallyunderstood by those skilled in the art. In the event that a conflictarises between an art-understood definition of a word or phrase and adefinition of the word or phrase as specifically taught in thisspecification, the specification shall control. Headings used herein aremerely for convenience, and are not to be construed as limiting in anyway.

[0066] Standard reference works setting forth the general principles ofrecombinant DNA technology known to those of skill in the art includeAusubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons, New York, 1998 Molecular Cloning: A Laboratory Manual (3rd ed.)Sambrook, J. & D. Russell, Eds.Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2001); Kaufman et al., Eds., HANDBOOK OF MOLECULARAND CELLULAR METHODS IN BIOLOGY AND MEDICINE, CRC Press, Boca Raton,1995; McPherson, Ed., DIRECTED MUTAGENESIS: A PRACTICAL APPROACH, IRLPress, Oxford, 1991.

[0067] As used herein, the term “microsatellite” refers to a geneticlocus comprising a short (e.g., 1-5 nucleotide), tandemly repeatedsequence motif. As used herein “mononucleotide microsatellite” refers toa genetic locus comprising a repeated nucleotide (e.g., C/G).“Dinucleotide microsatellite” refers to genetic locus comprising a motifof two nucleotides that is tandemly repeated (e.g., GC/GC).“Trinucleotide microsatellite” refers to a genetic locus comprising amotif of three nucleotides that is tandemly repeated (e.g., CAG/CTG,CGA/TCG, CGG/CCG). “Tetranucleotide microsatellite” refers to a geneticlocus comprising a motif of four nucleotides that is tandemly repeated(e.g., TGCC/GGCA). “Pentanucleotide microsatellite” refers to a geneticlocus comprising a motif of five nucleotides that is tandemly repeated(e.g., CCCCT/AGGGG). Microsatellites may contain repeat-motifinterspersions, or “cryptically simple sequence” (Tautz, D. et al.(1986) Nature 322(6080):652-656). Such repeat-motif interspersionsinclude simple repeat-motif interspersions wherein the microsatellitecontains one or more interspersed repeats with the same length as thetandemly repeated sequence motif, but a different repeat sequence(Eichler, E. E. et al. (1994) Nat. Genet. 8:88-94; Eichler, E. E. et al.(1996) Hum. Mol. Genet. 5:319-330). For example, if the tandemlyrepeated sequence motif is TGCC, a simple repeat-motif interspesion mayappear as follows: TGCC(TCTG)₂(TGCC)₃, wherein the interspersed repeat“TCTG” interrupts the repeat of the TGCC tandemly repeated sequencemotif. Repeat-motif interspersions also include more complexrepeat-motif interspersions wherein the repeat motif interspersion isnot the same length as the tandemly repeated sequence motif. Forexample, if the tandemly repeated sequence motif is TGCC, a complexrepeat-motif interspersion may appear as follows:(TGCC)₃TG(TGCC)₃TGC(TGCC)₂, wherein the tandemly repeated sequence motifis interrupted by TG and TGC. Other more complex repeat motifinterspersions include the combination of the simple repeat-motifinterspersion and the complex repeat-motif interspersion in the samemicrosatellite. For example, such a complex sequence repeat-motifinterspersion may appear as follows:(TGCC)_(n)(TCTG)_(o)(TGCC)₃TG(TGCC)₃TGC(TGCC)₂TGCCC(TGCC)_(p), whereinboth forms of interspersed repeats interrupt the tandemly repeatedsequence motif, TGCC. Microsatellites with and without interspersedrepeats are encompassed by the term “microsatellites” as used herein.

[0068] As used herein, the term “stutter” or “stutter signal” refers toa PCR artifact wherein microsatellites are incorrectly amplified suchthat a diverse population of fragments of varying length are producedfor each allele in the genomic source DNA. A typical “stutter signal”results from one or more PCR products that differ from the appropriatelength of the microsatellite-containing fragment by one or morerepeat-unit lengths of the microsatellite. As used herein “appropriatelength of the microsatellite-containing fragment” refers to the lengthpredicted from the primer sequences and the genomic target sequence,with or without one added nucleotide, depending on whether the PCRconditions promote or suppress the polymerase terminal transferase sidereaction. A stutter signal may be perceived with the naked eye, such asby examining a band on an agarose or polyacrylamide gel, or may beperceived with the aid of instrumentation. A stutter signal seen on agel typically appears as a blurry, shadow band due to microsatelliteswhich are incorrectly amplified such that a diverse population offragments of varying length are produced. A stutter signal as detectedon a GeneScan trace or other electropherogram may appear as a quantifiedsignal, such as a peak on a graph. Stutter signals may be represented byany means, such as, but not limited to brightness, intensity (e.g.,maximum intensity), magnitude of a signal output (e.g., peak height,integration of the area of a peak, peak width at half peak height), andthe like. For example, in a GeneScan trace for a tetranucleotidemicrosatellite, a major stutter signal is typically seen as a peak foundat four nucleobase units downfield (i.e., at a location corresponding toa shorter fragment) on the electropherogram from the major peak, whichrepresents the allele. Other, less prominent stutter signals may befound at 8 and 12 nucleobase units downfield on the chromatogram. Asused herein, the term “reducing stutter” is intended to mean theproduction of lower amounts of amplified stutter product, as reflectedby a decrease in the number or signal intensity of stutter fragments.

[0069] As used herein “sorbitol” refers to the polyol (polyhydricalcohol) corresponding to glucose, represented by the followingstructural formula:

[0070] As used herein, the term “isolated nucleic acid molecule” refersto a nucleic acid molecule (DNA or RNA) that has been removed from itsnative environment.

[0071] As used herein, “DNA” refers to deoxyribonucleic acid in itsvarious forms as understood in the art, such as genomic DNA, cDNA,isolated nucleic acid molecules, vector DNA, chromosomal DNA. “Nucleicacid” refers to DNA or RNA in any form. Examples of isolated nucleicacid molecules include, but are not limited to, recombinant DNAmolecules contained in a vector, recombinant DNA molecules maintained ina heterologous host cell, partially or substantially purified nucleicacid molecules, and synthetic DNA molecules. Typically, an “isolated”nucleic acid is free of sequences which naturally flank the nucleic acid(i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) inthe genomic DNA of the organism from which the nucleic acid is derived.Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule,is generally substantially free of other cellular material or culturemedium when produced by recombinant techniques, or of chemicalprecursors or other chemicals when chemically synthesized.

[0072] As used herein “nucleobase sequence” refers to a sequence ofconsecutive nucleobases.

[0073] As used herein, “anneal” refers to specific interaction betweenstrands of nucleotides wherein the strands bind to one anothersubstantially based on complementarity between the strands as determinedby Watson-Crick base pairing. It is not necessary that complementaritybe 100% for annealing to occur.

[0074] As used herein, “amplifying” refers to enzymatically increasingthe amount of a specific nucleotide sequence in a polymerase chainreaction.

[0075] As used herein “incubating” refers to a maintaining a state ofcontrolled conditions such as temperature over a period of time.

[0076] As used herein “denaturation” refers to the separation ofnucleotide strands from an annealed state. Denaturation may be inducedby a number of factors including ionic strength of the buffer,temperature, or chemicals that disrupt base pairing interactions.

[0077] As used herein “G+C content” refers to the relative amount ofguanosine and cytosine present in a given nucleic acid or portionthereof that is of interest, such as a microsatellite. The “G+C content”of a given nucleobase sequence, expressed in percent, can be calculatedfrom the formula 100(#G+#C)/Tot where #G is the number of guaninenucleobases in the nucleobase sequence, #C is the number of cytosinenucleobases in the nucleobase sequence, and Tot is the total number ofnucleobases in the nucleobase sequence.

[0078] As used herein, “sufficient amount of time” when referring totime for the amplification of nucleic acid, refers to the time whichallows the enzyme used to complete the polymerization of deoxynucleotidetriphosphates into the amplifying nucleic acid. The amount of timerequired varies depending on several factors which are well-known bypersons of ordinary skill in the art. General principles of PCR andstrategies for amplification may be found in such texts as, for example,Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons, New York, 2001 and THE POLYMERASE CHAIN REACTION, Mullis, K. B.,F. Ferre, and R. A. Gibbs, Eds., Birkhauser, Boston, 1994; AND MOLECULARCLONING: A LABORATORY MANUAL (3rd ed.) Sambrook, J. & D. Russell, Eds.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).

[0079] As used herein “conditions sufficient to amplify microsatellites”refers to reaction conditions for the PCR reactions. The reactionconditions include the chemical components of the reaction and theirconcentrations, the temperatures used in the reaction cycles, the numberof cycles of the reaction, and the durations of the stages of thereaction cycles.

[0080] Typically, buffered water is used as the solvent for thereaction. The other chemical components of standard PCR reactionsinclude a DNA polymerase, deoxyribonucleoside triphosphates (“dNTPs”),oligonucleotide primers, divalent metal ion, and a DNA sample expectedto contain the PCR target.

[0081] The solvent used for PCR typically contains a buffering agentsuch as Tris-HCl and non-buffering salts such as KCl. The bufferingagent may be any known buffers in the art, and may be varied to optimizePCR results by routine experimentation. Persons of ordinary skill in theart will readily be able to determine optimal buffering conditions. SomePCR buffers may be optimized depending on the enzyme used. As anexample, but not by way of limitation, AmpliTaq Gold® DNA polymerase hasan optimum KCl concentration of 50 mM, AmpliTaq® DNA Polymerase, Stoffelfragment has an optimum KCl concentration of 10 mM, and rTth DNAPolymerase and rTth DNA Polymerase XL, have an optimum KCl concentrationof 75-100 mM.

[0082] Divalent metal ions are often advantageous to allow thepolymerase to function efficiently. For example, but not by way oflimitation, magnesium ion allows certain DNA polymerases to functioneffectively. Typically, MgCl₂ or MgSO₄, is added to reaction buffers tosupply the optimum magnesium ion concentration. The magnesium ionconcentration required for optimal PCR amplification may depend on thespecific set of primers and template used. Thus, the amount of magnesiumsalt added to achieve optimal amplification is often determinedempirically, and is a routine practice in the art. Generally, theconcentration of magnesium ion for optimal PCR can vary between 1 and 10mM. A typical range of magnesium ion concentration in PCR reactions isbetween 1.0 and 4.0 mM, varying around a midpoint of 2.5 mM.

[0083] Deoxynucleotide triphosphates (“dNTPs”), which are the buildingblocks of the amplifying nucleic acid molecules, are typically suppliedin standard PCR reactions at a concentration of 40-200 μM each ofdeoxyadenosine triphosphate (“dATP”), deoxyguanosine triphosphate(“dGTP”), deoxycytidine triphosphate (“dCTP”) and thymidine triphosphate(“dTTP”). Other dNTPs, such as deoxyuridine triphosphate (“dUTP”), anddNTP analogs, and conjugated dNTPs may also be used, and are encompassedby the term “dNTPs” as used herein. While use of dNTPs at suchconcentrations is amenable to the methods of the invention,concentrations of dNTPs higher than 200 μM may be advantageous. Thus, insome embodiments of the methods of the invention, the concentration ofeach dNTP is generally at least 500 μM and may range up to 2 mM. In somefurther embodiments, concentration of each dNTP may range from 0.5 mM to1 mM.

[0084] The enzyme that polymerizes the nucleotide triphosphates into theamplified fragments of the PCR may be any DNA polymerase, includingheat-resistant polymerases known in the art. Polymerases that may beused in the invention include, but are not limited to DNA polymerasesfrom such organisms as Thermus aquaticus, Thermus thermophilus,Thermococcus litoralis, Bacillus stearothermophilus, Thermotoga maritimaand Pyrococcus ssp. The enzyme may be isolated from the source bacteria,produced by recombinant DNA technology or purchased from commercialsources. For example, DNA polymerases are available from AppliedBiosystems and include AmpliTaq Gold® DNA polymerase; AmpliTaq® DNAPolymerase; AmpliTaq® DNA Polymerase, Stoffel fragment; rTth DNAPolymerase; and rTth DNA Polymerase XL. Other suitable polymerasesinclude, but are not limited to Tne, Bst DNA polymerase large fragmentfrom Bacillus stearothermophilus, Vent and Vent Exo- from Thermococcuslitoralis, Tma from Thermotoga maritima, Deep Vent and Deep Vent Exo-and Pfu from Pyrococcus, and mutants, variants and derivatives of theforegoing.

[0085] Oligonucleotide primers are added to the reaction and demarcatethe 5′ and 3′ ends of the amplified fragment. One oligonucleotide primeranneals to the sense (+strand) of the denatured, template DNA, and theother oligonucleotide primer anneals to the antisense (−strand) of thedenatured, template DNA. Typically, oligonucleotide primers are 12-25nucleotides in length, however, they may be shorter or longer dependingon the specific template sequence to be amplified, and the length of theprimer is not essential to the operation of the invention.Oligonucleotide primers may be designed to anneal to specific portionsof DNA that flank a microsatellite of interest to specifically amplifythe portion of DNA between the primer-complementary sites. Generally,oligonucleotide primers are chemically synthesized. One of ordinaryskill in the art may easily design specific primers to amplify a targetmicrosatellite of interest. Furthermore, there are many known primersequences to amplify microsatellite regions. Any of these may be used,and are within the scope of the invention.

[0086] The oligonucleotide primers may be composed of adenosine,thymidine, guanosine, cytidine, uracil, nucleoside analogs (e.g., lockednucleic acids (LNA), peptide nucleic acid (PNA), phosporamidites) andnucleosides containing or conjugated to chemical moieties such asradionuclides (e.g., ³²P, ³⁵S), fluorescent molecules, minor groovebinders, or any other nucleoside conjugate known in the art.

[0087] In some embodiments of the invention, a fluorophore is used totag at least one primer of the PCR reaction. In some embodiments primersfor different target fragments can be tagged with different fluorophores(that produce differently colored products) and may be used in the samemultiplex PCR reaction and subsequently analyzed together. Typically,the forward primer is tagged, but the reverse primer may also be tagged.Examples of fluorophores include, but are not limited to, fluorescein(which absorbs maximally at 492 nm and emits maximally at 520 nm);TAMRA, N,N,N′,N′-tetramethyl-6-carboxyrhodamine (which absorbs maximallyat 555 nm and emits maximally at 580 nm); FAM, 5-carboxyfluorescein(which absorbs maximally at 495 nm and emits maximally at 525 nm); JOE,2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (which absorbsmaximally at 525 nm and emits maximally at 555 nm), ROX,6-carboxy-X-rhodamine (which absorbs maximally at 585 nm and emitsmaximally at 605 nm); CY3 (which absorbs maximally at 552 nm and emitsmaximally at 570 nm), CY5 (which absorbs maximally at 643 nm and emitsmaximally at 667 nm); TET, tetrachloro-fluorescein (which absorbsmaximally at 521 nm and emits maximally at 536 nm); and HEX,hexachloro-fluorescein (which absorbs maximally at 535 nm and emitsmaximally at 556 nm).

[0088] Other known components of PCR reactions may be used within thescope of the invention. Such components include, but are not limited to,detergents (e.g., Triton X-100, Nonidet P-40 (NP-40), Tween-20) andagents that disrupt mismatching of nucleotide pairs, such asdimethylsulfoxide (DMSO), and tetramethylammonium chloride (TMAC).

[0089] PCR reaction times, temperatures and cycle numbers may be variedto optimize a particular reaction as a matter of routineexperimentation. Those of ordinary skill in the art will recognize thefollowing as guidance in determining the various parameters for PCRreactions, and also will recognize that variation of one or moreconditions is within the scope of the invention.

[0090] PCR reaction temperature and time are determined in three stages:denaturation, annealing and extension. One round of denaturation,annealing and extension is referred to as a “cycle.” Denaturation isgenerally conducted at a temperature that permits the strands of DNA toseparate, yet not destroy polymerase activity. Generally,thermoresistant polymerases are used. However, heat-labile polymerasesmay be used if they are replenished after each denaturation step of thePCR. Thermoresistant polymerases can withstand high temperatures andmaintain some level of activity. Typically, denaturation is conductedabove 90° C. and below 100° C. In some embodiments, denaturation isconducted at a temperature of 94-95° C. Denaturation of DNA is generallyconducted for at least 1 to 30 seconds. In some embodiments,denaturation is conducted for 1 to 15 seconds. In other embodiments,denaturation is conducted for up to 1 minute or more. In addition to thedenaturation of DNA, for some polymerases, such as AmpliTaq Gold®,incubation at the denaturation temperature also serves to activate theenzyme. Therefore, it may be advantageous to allow the first step of PCR(denaturation) to be longer than subsequent denaturation steps whenthese enzymes are used.

[0091] During the annealing phase, oligonucleotide primers anneal to thetarget DNA in their regions of complementarity and are substantiallyextended by the DNA polymerase once the latter has bound to theprimer-template duplex.

[0092] In a conventional PCR, the annealing temperature typically is ator below the melting point (T_(m)) of the least stable primer-templateduplex, where T_(m) can be estimated by any of several theoreticalmethods well known to practitioners of the art. For example, the T_(m)may be determined by the formula:

[0093] T_(m)=(4° C.×number of G and C bases)+(2° C.×number of A and Tbases) Typically, in standard PCRs, the annealing temperature is 5° C.to 10° C. below the estimated T_(m) of the least stable primer-templateduplex. The annealing time is between about 30 seconds and 2 minutes.However, in certain embodiments of the methods of the invention, thehigh concentration of sorbitol increases reagent viscosity and appearsto slow certain steps of the reaction (e.g., primer annealing andpolymerase binding to the primer-template duplex). Thus, in certainembodiments of the methods of the invention, the annealing step isperformed for a longer period of time than would be used in standard PCRprotocols, typically for at least 3 minutes and as long as 5 to 6minutes.

[0094] Sorbitol not only increase reaction viscosity, but is also a mildDNA denaturant. Thus, in certain embodiments of the methods of theinvention, it is may be advantageous to use a lower temperature forannealing primers to the template than would be used by one of ordinaryskill in the art for standard PCR reactions. In general, temperatureslower than 10° C. below the T_(m) (estimated in the absence of additive)may be employed in certain embodiments of the invention.

[0095] The annealing phase typically is followed by an extension phase.“Extension” is conducted for a sufficient amount of time to allow theenzyme to complete primer extension into the appropriately sizedfragments. As discussed above, the addition of high concentrations ofsorbitol increases the viscosity of the reaction, makingunconventionally long extension times advantageous in certainembodiments of the methods of the invention; i.e., the use of extensiontimes that are longer compared to extension times one of ordinary skillin the art would calculate for standard PCR reactions. Furthermore, asnoted above for the annealing phase, sorbitol is a mild denaturant.Thus, in some embodiments of the methods of the invention, it may beadvantageous to also use a lower temperature for extension than would beused by one of ordinary skill in the art for standard PCR reactions.Thus, for some embodiments, temperatures for extension are below thetemperature reported for optimal activity of the polymerases used.

[0096] The number of cycles of PCR (denaturation, annealing andextension) used will determine the desired amount of amplification. PCRis an exponential amplification of DNA molecules. Thus, theoretically,after each cycle of PCR, there are twice the number of fragments thatwere present in the previous cycle. Typically, 20-30 cycles of PCR areperformed. More typically, 25-30 cycles are performed, although cyclenumber is not particularly limited.

[0097] For some embodiments, it is advantageous to incubate thereactions at a certain temperature following the last phase of the lastcycle of PCR. In some embodiments, a prolonged extension phase isselected. In other embodiments, an incubation at a low temperature(e.g., 4° C.) is selected.

[0098] In one embodiment of the invention, methods are provided forreducing stutter in the amplification of a microsatellite wherein asample containing a microsatellite of interest is provided, wherein themicrosatellite has a G+C content of greater than 50%. The sample iscontacted with at least one enzyme having nucleic acid polymeraseactivity, and the sample is incubated with the enzyme for a sufficientamount of time and under conditions sufficient to amplify saidmicrosatellite. The incubation is performed in the presence of an amountof sorbitol that is effective to reduce stutter relative to the amountof stutter observed in the absence of sorbitol. The PCR reactionincludes primers that are selected to amplify the target microsatelliteof interest and which are optionally tagged, dNTPs, buffer, the samplecontaining the template nucleic acid to be amplified, sorbitol and thepolymerase.

[0099] In another embodiment of the invention, primer extensionreactions may be performed with greater accuracy when conducted in thepresence of sorbitol. In primer extension reactions, an oligonucleotideprimer is permitted to bind to a specific target sequence on a nucleicacid molecule in the presence of dNTPs and a polymerase. The reaction isincubated and the polymerase polymerizes the dNTPs and extends theoligonucleotide primer in the 5′ to 3′ direction to form the complementof the target nucleic acid molecule. The primer may contain a detectablelabel, or the dNTPs used may contain a detectable label. Further, thedNTPs may be modified dNTPs, including, such as, but not limited to,dideoxynucleotide triphosphates.

[0100] The microsatellites amplified in the methods of the invention maybe mononucleotide microsatellites, dinucleotide microsatellites,trinucleotide microsatellites, tetranucleotide microsatellites, andpentanucleotide microsatellites, and the microsatellites may includerepeat motif interspersions. Mononucleotide microsatellites can be arepeat of C. The complementary strand of this portion of DNA wouldcontain repeats of G. To denote the microsatellite with itscomplementary strand, the notation C/G is used. In some embodiments, themicrosatellite is a dinucleotide microsatellite. Examples ofdinucleotide microsatellites include, CG/CG. In other embodiments, themicrosatellite is a trinucleotide microsatellite, including but notlimited to CAG/CTG, CGA/TCG and CGG/CCG. In still other embodiments, themicrosatellite is a tetranucleotide microsatellite, including, but notlimited to TGCC/GGCA. Examples of loci that contain such tetranucleotidemicrosatellites include, but are not limited to D2S1338 (TGCC)(TTCC);and D7S809 (AGGA)(AGGC). In still other embodiments, the microsatelliteis a pentanucleotide microsatellite, including, but not limited toCCCCT/AGGGG.

[0101] The microsatellites amplified in the method of the inventiongenerally have a G+C content of greater than 50%. In some embodiments,the microsatellite has a G+C content of 66% or more. In otherembodiments, the microsatellite has a G+C content of 75% or more. Inother embodiments, the microsatellite has a G+C content of 100%.

[0102] The amount of sorbitol added to the PCR reaction is generally inan amount effective to reduce stutter relative to the amount of stutterobserved in the absence of sorbitol. In some embodiments, the amount ofsorbitol added is 1.5 to 3.5 M. In some embodiments, sorbitol is addedin an amount of 2.0 to 3.0 M. In other embodiments, sorbitol is added inan amount of 2.0 M. The sorbitol may be added from a separate stock ormay be added as part of another PCR reagent. For example, in oneembodiment of the invention, the sorbitol is included in the DNApolymerase preparation and is added when the enzyme is added to thereaction. In another embodiment of the invention, the sorbitol isincluded with a preparation of magnesium ion containing reagent (e.g.,MgCl₂ or MgSO₄) so that the sorbitol is added with the MgCl₂ or MgSO₄.

[0103] Sorbitol has chemical properties that may be exploited forvarious purposes. For example, sorbitol is rather hydrolytically inert,which may impart-a longer shelf-life to the PCR reagents. As anotherexample, sorbitol possesses no acid-base properties, so it does notaffect the pH of the PCR reaction when added in high concentrations.

[0104] In accordance with the embodiments of the methods of theinvention, the addition of sorbitol to the reactions is effective inreducing stutter to between 90% and 20% of the amount of stutterobtained in the absence of sorbitol. In some embodiments, the amount ofstutter observed with the addition of sorbitol is reduced to 90% or lessthan the amount observed in the absence of sorbitol. In someembodiments, the amount of stutter observed with the addition ofsorbitol is reduced to 80% or less than the amount observed without theaddition of sorbitol. In other embodiments, the amount of stutterobserved with the addition of sorbitol is reduced to 70% or less thanthe amount observed without the addition of sorbitol. In otherembodiments, the amount of stutter observed with the addition ofsorbitol is reduced to 60% or less than the amount observed without theaddition of sorbitol. In other embodiments, the amount of stutterobserved with the addition of sorbitol is reduced to 50% or less thanthe amount observed without the addition of sorbitol. In otherembodiments, the amount of stutter observed with the addition ofsorbitol is reduced to 40% or less than the amount observed without theaddition of sorbitol. In other embodiments, the amount of stutterobserved with the addition of sorbitol is reduced to 30% or less thanthe amount observed without the addition of sorbitol. The reduction ofstutter is measured by determining the percent stutter in the presenceof sorbitol (+S), and in the absence of sorbitol (−S), and performingthe following calculation (+S)/(−S)×100. Percent stutter is determined,for example, by the peak height of the stutter signal over the peakheight of the allele signal times 100. Alternatively, percent stutter isdetermined by peak width at half peak height of the stutter signal overthe peak width at half peak height of the allele signal times 100. Inother embodiments, percent stutter is determined by area of the peak ofthe stutter signal over area of the peak of the allele signal times 100.Any other method of determining percent stutter in the presence ofsorbitol relative to the percent stutter in the absence of sorbitol maybe used, and the percent reduction determined.

[0105] In further embodiments of the invention, methods are provided inwhich a sample containing nucleic acid that is suspected of containingone or more microsatellites (e.g., mononucleotide microsatellites,dinucleotide microsatellites, trinucleotide microsatellites, and/ortetranucleotide microsatellites having a G+C content of greater than50%) is contacted with an enzyme that polymerizes nucleotides in thepresence of an effective amount of sorbitol to reduce observed stutterrelative to the amount of stutter observed in the absence of sorbitol,and amplifying at least one nucleobase sequence containing at least onemicrosatellite of the nucleic acid contained in the sample.

[0106] In further embodiments of the invention, PCR reactions asdescribed herein are employed to amplify fragments from at least onemicrosatellite region. In certain embodiments, the fragments areamplified with a detectable tag (e.g., a fluorophore-tagged primer) orwith a hybridization enhancer (e.g., a minor groove binder). Where morethan one microsatellite region is to be amplified, detectable tags areselected such that different products are easily distinguished. As anexample, but not by way of limitation, different colored fluorophoresmay be used to amplify different microsatellites. Furthermore, the samecolor fluorophore may be used to amplify fragments containingmicrosatellites that generate fragments of different sizes which arereadily discernable, e.g., by electrophoretic separation. The PCRproducts can be analyzed in on a sieving or non-sieving medium. In someembodiments of the invention, for example, the PCR products are analyzedby capillary electrophoresis as described in Wenz, H. et al. (1998)Genome Res. 8:69-80. In other embodiments of the invention, for example,the PCR products are analyzed by slab gel electrophoresis as describedin Christensen, M. et al. (1999) Scand. J. Clin. Lab. Invest. 59(3):167-177. Fragments may be analyzed by chromatography, e.g., sizeexclusion chromatography (SEC).

[0107] In certain embodiments, the methods of the invention decrease thenumber or intensity of stutter bands, or shadow bands, on standard gels.Thus, the methods of the invention provide for more simplified and moreaccurate interpretation of banding patterns on gels, with increasedresolution of bands. Accordingly, for some embodiments of the invention,the PCR reactions may be analyzed on agarose or polyacrylamide gelsusing standard techniques.

[0108] In accordance with the embodiments of the invention, the use ofsorbitol in the analysis of microsatellites by PCR provides a markedenhancement in the diagnostic capabilities of microsatellites in mono-,di-, tri-, tetra- and pentanucleotide repeat microsatellites.

[0109] In some embodiments, the invention provides kits. In someembodiments, the kits of the invention contain a concentrated stocksolution of sorbitol for addition to the PCR reactions such thatsorbitol is diluted to be present in an amount of 1.5 to 3.5 M in thePCR. Typically, the sorbitol is diluted to be present in an amount of 2to 3 M. In addition to the sorbitol solution, the kits may contain atleast one of the following reagents: dNTPs (either separately or as amixture), DNA polymerase, buffer, and primers to amplifymicrosatellites. The kits may also contain conventional kit components,such as instructions for use.

[0110] Microsatellite markers that exhibit a high degree of polymorphismare exploited in many important applications. Recently, the A-repeat BATloci have been used in studying microsatellite instability (MSI) whichcorrelates with certain cancers (Chen et al. (1995) Cancer Res.55:174-180; Ionov et al. (1993) Nature 363:558-561 De La Chapelle (1999)Eur. J. Hum. Genet. 7:407-408). However, due to the small allelic sizedifferences in these markers and stutter during PCR, it has beendifficult to determine whether the DNA samples are homozygous orheterozygous. Additionally, polymorphic variations at these loci inindividuals of different ethnic origins supports the need to define thedifferent allelic profiles and frequencies (Pyatt et al. (1999) Am. J.Pathol. 155:349-353).

[0111] Detection of genetic disorders associated with aberrantmicrosatellites is an application of the methods of the invention.Samples may be taken from tissues or individuals suspected of harboringaberrant microsatellites in their DNA and the DNA may be amplified byPCR in the presence of an sufficient amount of sorbitol under conditionssufficient to reduce stutter relative to observed stutter in the absenceof sorbitol for microsatellites with a G+C content of greater than 50%.The resulting PCR products may be compared to PCR products from normaltissue, or tissue from normal individuals, and variation assessed.Aberrant microsatellites may indicate a propensity to develop agenetically-based disorder, or may indicate the presence of agenetically-based disease. Such disorders include, but are not limitedto cancer, pre-cancerous conditions (e.g., chronic lymphocytic leukemia)and genetic disorders such as, but not limited to the spinocerebellarataxias, Huntington disease, oculopharyngeal muscular dystrophy,myotonic dystrophy, and Fragile X Syndrome. As an example, but not byway of limitation, the fragile X syndrome may be diagnosed using themethod of the invention by amplification, in the presence of asufficient amount of sorbitol, the FRAXA locus, which has an unstableand pathogenic CGG/CCG trinucleotide repeat.

[0112] In a specific embodiment, PCR amplification reactions may be setup to amplify a trinucleotide repeat using the primers to amplify withinthe FRAXA locus. The sense primers of each of the loci may be taggedwith a fluorophore. The PCR reactions may be set up as follows: 50 mMKCl, 10 mM Tris-HCl (pH=9.0), 0.1% Triton X-100, 4.5 mM MgCl₂, 1.0 mMeach of dATP, dGTP, dCTP, and dTTP, 2.5 U Taq DNA polymerase and 2 Msorbitol. The reaction may then proceed as follows: 94° C. for 1minutes, and 28 cycles of 94° C. for 15 seconds, 40° C. for 3 minutesand 65° C. for 5 minutes.

[0113] PCR reactions are then analyzed by denaturing samples andseparating using a capillary gel electrophoresis protocol and using anABI PRISM® 310 genetic analyzer, or by separating on a 4.5%, 29:1acrylamide:bis acrylamide, 8 M urea gel prepared for an ABI 377Automated Fluorescence DNA Sequencer. Fragment analysis may be analyzedwith GeneScan Software from Applied Biosystems. Alteration in the sizeof the amplified fragment as compared to normal, control tissues orsamples could be indicative of Fragile X Syndrome.

[0114] The methods of the invention are also useful in such applicationsas genetic mapping (linkage analysis). Linkage analysis may beaccomplished, for example, by using a panel of primers to amplify a setof loci containing microsatellites that have a G+C content of greaterthan 50% in the presence of a sufficient amount of sorbitol to reducethe observation of stutter from that observed in the absence ofsorbitol. Genetic loci such as D2S1338 and D7S809, for example, may beamplified for a given sample of DNA from an individual. Theamplification may be done in the same reaction if the primers aredifferentially labeled (such as by using fluorophore tags) that willallow ready identification of PCR products following amplification.

[0115] In the field of human identity, tetranucleotide microsatellitesare used in forensic casework, establishment of convicted felondatabases, disaster and military victim identification (Fre'geau et al.(1993) Biotechniques 15:100-119). Furthermore, they have proved usefulin forensics to identify human remains (Hagelberg et al. (1991) Nature352:427429; Hammond et al. (1994) Am. J. Hum. Genet. 55:175-189). In theanalysis of museum specimens (Ellegren et al. (1991) Nature 354:113) andin parentage testing. Tetranucleotide microsatellites are specificallypowerful in these applications, since multiple microsatellite tests thathave matching probabilities of one in several billion individuals arenow available. Examples of microsatellite containing alleles which canbe used for paternity, forensic and other personal identification areD2S1338 ((TGCC)_(n)(TTCC)_(n)), and D7S809.

[0116] Also of special value in the identification of individuals is thePCR analysis of the highly polymorphic D loop of mitochondrial DNA,which contains a polymorphic C/G mononucleotide repeat; this analysistypically includes Sanger sequencing of the PCR product.

[0117] Personal identification tests may be performed on any specimenthat contains nucleic acid such as bone, hair, blood, tissue and thelike. DNA may be extracted from the specimen and a panel of primers toamplify a set of microsatellites used to amplify DNA in the presence ofan effective amount of sorbitol to reduce stutter from the specimen togenerate a set of amplified fragments. In forensic testing, thespecimen's microsatellite amplification pattern is compared with a knownsample the presumptive victim (the presumed matching source) or iscompared to the pattern of amplified microsatellites derived from thepresumptive victim's family members (e.g., the mother and father)wherein the same set of microsatellites is amplified in the presence ofan effective amount of sorbitol to reduce stutter using the sameprimers. The pattern of microsatellite amplification may be used toconfirm or rule out the identity of the victim. In paternity testing,the specimen is generally from the child and the comparison is made tothe microsatellite pattern from the presumptive father, and may includematching with the microsatellite pattern from the child's mother. Thepattern of microsatellite amplification may be used to confirm or ruleout the identity of the father. The panel may include microsatelliteswith a G+C content of greater than 50% such as, for example, D2S1338 andD7S809. PCR conditions include 5-10 ng genomic DNA, 10 pmoles eachfluorophore-tagged primer, 2.5 M sorbitol, 1 mM each dNTPs, 5 mM MgCl₂,50 mM KCl, 10 mM Tris-HCl, 5 U DNA polymerase. PCR cycle conditions maybe 1 min 94° C., followed by 30 cycles of 20 seconds at 94° C., 3minutes at 50° C., 3 minutes at 60° C., followed by one cycle of 10minutes at 60° C. The products are examined by capillary gelelectrophoresis coupled with GeneScan 310 analysis.

[0118] Dinucleotide microsatellites are also used in paternity testingfor cattle, dogs, horses and other animals (Primmer et al. (1995) Mol.Ecol. 4:493-498). In a clinical setting, microsatellite markers can beused to monitor the degree of donor engraftment in bone marrowtransplants. In hospitals, microsatellite markers are useful in specimenmatching tracking. More recently, microsatellite markers have alsoentered other fields of science such as population biology studies onhuman racial and ethnic group differences (Goldstein et al. (1995) Proc.Natl. Acad. Sci. USA 92:6723-6727) and on variation in animal and planttaxa (Bruford et al. (1993) Curr. Biol. 3:939-943).

[0119] Reduction in stutter bands in accordance with the presentinvention is useful in all of the above applications, which areillustrative and not limiting, because, inter alia, the interpretationof the data is facilitated by the method of the invention. The methodsof the invention may be used in conjunction with the methods describedin the references cited herein, the disclosure of each of which isincorporated herein by reference in its entirety. In particular, themethods of the invention will simplify analyses of forensic samples, andtherefore find particular utility in the forensic field.

[0120] The invention will be further described using the followingactual examples, which are merely illustrative of some embodiments ofthe invention. The examples should not be construed in any way to limitthe scope of the invention, which is defined by the appended claims.

EXAMPLES Example 1

[0121] PCR reactions were used to amplify the DNA tetranucleotidemicrosatellite, D2S1338 ((TGCC)(TTCC)) in the presence or absence ofsorbitol (FIG. 1). The 50 ul reactions contained 20 mM Tris, 20 mMammonium sulfate, 4.5 mM magnesium sulfate 1 mM each or dATP, dGTP, dCTPand dTTP, 12 pmoles oligonucleotide primers, 2 ng human genomic DNAtemplate, 5U AmpliTaq Gold® DNA polymerase in water. In reactions inwhich sorbitol was added, the reactions contained 2.0 M sorbitol. Thereaction conditions were as follows: 95° C. for 11 minutes, followed by28 cycles of 94° C. for 30 seconds, 55° C. for 4 minutes and 69° C. for6 minutes, followed by incubation at 60° C. for 45 minutes. FollowingPCR, samples were mixed with ROX fluorescent size marker (AppliedBiosystems). Samples were denatured and separated using a protocol forcapillary electrophoresis prepared for an ABI 310 Automated GeneticAnalyzer according to the manufacturer's specifications. Fragmentanalysis data was analyzed with GeneScan Software from AppliedBiosystems. For each set of peaks observed (see FIG. 1), each peakcorresponds to a fluorescence labeled, single stranded DNA molecule. Theposition of the peak on the x axis corresponds to the length of the DNAmolecule. The height of each peak corresponds to the relative amount ofPCR product for this length, as determined by relative fluorescenceintensity measured in arbitrary units. A stutter peak for thetetranucleotide repeat is found four peaks to the left of the main peak(where the “main peak” is the allele). A reduction in the height of thestutter peak relative to the main peak is indicative of a reduction instutter. When measuring stutter reduction, the height of the stutterpeak over the height of the main peak×100 provides the percent stutter.If there are multiple stutter peaks, they are not taken collectively.Rather the height of the main stutter peak over the height of the allelepeak×100 gives the percent stutter. The results for the reactions withsorbitol, and control samples are shown in FIG. 1 and are summarized inTable 1: TABLE 1 Results of sorbitol on amplification of tetranucleotidemicrosatellites % Stutter Microsatellite Control + SorbitolTetranucleotide 8.3 5.1 (−39%)

[0122] The reference works, patents, patent applications, and scientificliterature, and other printed publications, including accession numbersto GenBank database sequences, that are referred to herein are herebyincorporated by reference in their entirety.

[0123] As those skilled in the art will appreciate, numerous changes andmodifications may be made to the embodiments of the invention withoutdeparting from the spirit of the invention. It is intended that all suchvariations fall within the scope of the invention.

What is claimed is:
 1. A method for reducing stutter in theamplification of a microsatellite comprising the steps of: (a) providinga sample comprising a microsatellite of interest, said microsatellitehaving a G+C content of greater than 50%; (b) contacting said samplewith at least one enzyme having nucleic acid polymerase activity; and(c) incubating said sample with said enzyme for a time and underconditions sufficient to amplify said microsatellite; wherein saidincubation is performed in the presence of an amount sorbitol effectiveto reduce said stutter relative to the amount of stutter observed in theabsence of sorbitol.
 2. A method for reducing stutter in theamplification of a mononucleotide microsatellite comprising the stepsof: (a) providing a sample comprising a microsatellite of interest, saidmicrosatellite having a G+C content of greater than 50%; (b) contactingsaid sample with at least one enzyme having nucleic acid polymeraseactivity; and (c) incubating said sample with said enzyme for a time andunder conditions sufficient to amplify said microsatellite; wherein saidincubation is performed in the presence of an amount of sorbitoleffective to reduce said stutter relative to the amount of stutterobserved in the absence of sorbitol.
 3. The method of claim 2 whereinsaid stutter is reduced to 60% or less the amount of stutter obtained inthe absence of sorbitol.
 4. The method of claim 2 wherein said sorbitolis present in an amount of 1.5 to 3.5 M.
 5. The method of claim 2wherein said sorbitol is present in an amount of 2.0 to 3.0 M.
 6. Amethod for reducing stutter in the amplification of a dinucleotidemicrosatellite comprising the steps of: (a) providing a samplecomprising a microsatellite of interest, said microsatellite having aG+C content of greater than 50%; (b) contacting said sample with atleast one enzyme having nucleic acid polymerase activity; and (c)incubating said sample with said enzyme for a time and under conditionssufficient to amplify said microsatellite; wherein said incubation isperformed in the presence of an amount of sorbitol effective to reducesaid stutter relative to the amount of stutter observed in the absenceof sorbitol.
 7. The method of claim 6 wherein said stutter is reduced to60% or less the amount of stutter obtained in the absence of sorbitol.8. The method of claim 6 wherein said sorbitol is present in an amountof 1.5 to 3.5 M.
 9. The method of claim 6 wherein said sorbitol ispresent in an amount of 2.0 to 3.0 M.
 10. A method for reducing stutterin the amplification of a trinucleotide microsatellite comprising thesteps of: (a) providing a sample comprising a microsatellite ofinterest, said microsatellite having a G+C content of greater than 50%;(b) contacting said sample with at least one enzyme having nucleic acidpolymerase activity; and (c) incubating said sample with said enzyme fora time and under conditions sufficient to amplify said microsatellite;wherein said incubation is performed in the presence of an amount ofsorbitol effective to reduce said stutter relative to the amount ofstutter observed in the absence of sorbitol.
 11. The method of claim 10wherein said microsatellite comprises a repeat selected from the groupconsisting of CAG/CTG, CCG/CGG, and CGA/TCG.
 12. The method of claim 10wherein said microsatellite has a G+C content of 66% or more.
 13. Themethod of claim 10 wherein said stutter is reduced to 60% or less theamount of stutter obtained in the absence of sorbitol.
 14. The method ofclaim 10 wherein said sorbitol is present in an amount of 1.5 to 3.5 M.15. The method of claim 10 wherein said sorbitol is present in an amountof 2.0 to 3.0 M.
 16. A method for reducing stutter in the amplificationof a tetranucleotide microsatellite comprising the steps of: (a)providing a sample comprising a microsatellite of interest, saidmicrosatellite having a G+C content of greater than 50%; (b) contactingsaid sample with at least one enzyme having nucleic acid polymeraseactivity; and (c) incubating said sample with said enzyme for a time andunder conditions sufficient to amplify said microsatellite; wherein saidincubation is performed in the presence of an amount of sorbitoleffective to reduce said stutter relative to the amount of stutterobserved in the absence of sorbitol.
 17. The method of claim 16 whereinsaid microsatellite comprises a TGCC/GGCA repeat.
 18. The method ofclaim 16 wherein said microsatellite has a G+C content of 66% or more.19. The method of claim 16 wherein said stutter is reduced to 60% orless the amount of stutter obtained in the absence of sorbitol.
 20. Themethod of claim 16 wherein said sorbitol is present in an amount of 1.5to 3.5 M.
 21. The method of claim 16 wherein said sorbitol is present inan amount of 2.0 to 3.0 M.
 22. A method for reducing stutter in theamplification of a pentanucleotide microsatellite comprising the stepsof: (a) providing a sample comprising a microsatellite of interest, saidmicrosatellite having a G+C content of greater than 50%; (b) contactingsaid sample with at least one enzyme having nucleic acid polymeraseactivity; and (c) incubating said sample with said enzyme for a time andunder conditions sufficient to amplify said microsatellite; wherein saidincubation is performed in the presence of an amount of sorbitoleffective to reduce said stutter relative to the amount of stutterobserved in the absence of sorbitol.
 23. The method of claim 22 whereinsaid microsatellite comprises a CCCCT/AGGGG repeat.
 24. The method ofclaim 22 wherein said microsatellite has a G+C content of 66% or more.25. The method of claim 22 wherein said stutter is reduced to 60% orless the amount of stutter obtained in the absence of sorbitol.
 26. Themethod of claim 22 wherein said sorbitol is present in an amount of 1.5to 3.5 M.
 27. The method of claim 22 wherein said sorbitol is present inan amount of 2.0 to 3.0 M.
 28. The method of claim 1 wherein saidmicrosatellite has a G+C content of 66% or more.
 29. The method of claim1 wherein said microsatellite has a G+C content of 75% or more.
 30. Themethod of claim 1 wherein said microsatellite has a G+C content of 100%.31. The method of claim 1 wherein said stutter is reduced to 90% or lessthan the amount of stutter obtained in the absence of sorbitol.
 32. Themethod of claim 1 wherein said stutter is reduced to 80% or less thanthe amount of stutter obtained in the absence of sorbitol.
 33. Themethod of claim 1 wherein said stutter is reduced to 70% or less thanthe amount of stutter obtained in the absence of sorbitol.
 34. Themethod of claim 1 wherein said stutter is reduced to 60% or less theamount of stutter obtained in the absence of sorbitol.
 35. The method ofclaim 1 wherein said stutter is reduced to 50% or less than the amountof stutter obtained in the absence of sorbitol.
 36. The method of claim1 wherein said stutter is reduced to 40% or less than the amount ofstutter obtained in the absence of sorbitol.
 37. The method of claim 1wherein said stutter is reduced to 30% or less than the amount ofstutter obtained in the absence of sorbitol.
 38. The method of claim 1wherein said incubation is performed in the presence of a set of dNTPs,said set comprising an amount of dNTP complementary to adenosine, anamount of dNTP complementary to guanosine, an amount of dNTPcomplementary to cytidine and an amount of dNTP complementary tothymine, wherein each of said amounts of dNTP is least 0.5 mM.
 39. Themethod of claim 1 wherein said incubation is performed in the presenceof a set of dNTPs, said set comprising an amount of dNTP complementaryto adenosine, an amount of dNTP complementary to guanosine, an amount ofdNTP complementary to cytidine and an amount of dNTP complementary tothymine, wherein each of said amounts of dNTP is least 1 mM.
 40. Themethod of claim 1 wherein said sorbitol is present in an amount of 1.5to 3.5 M.
 41. The method of claim 1 wherein said sorbitol is present inan amount of 2.0 to 3.0 M.
 42. A method comprising the steps of: (a)providing a sample comprising a nucleic acid containing one or moremicrosatellites selected from the group consisting of mononucleotidemicrosatellites, dinucleotide microsatellites, trinucleotidemicrosatellites, tetranucleotide microsatellites, and pentanucleotidemicrosatellites; and (b) amplifying at least one nucleobase sequence ofsaid nucleic acid, said nucleobase sequence comprising at least one ofsaid microsatellites; said amplified microsatellite having a G+C contentof greater than 50%; wherein said amplification is performed in thepresence of sorbitol.
 43. The method of claim 42 wherein the G+C contentof at least one of said amplified microsatellites is 66% or more. 44.The method of claim 42 wherein the G+C content of at least one of saidamplified microsatellites is 75% or more.
 45. The method of claim 42wherein the G+C content of at least one of said amplifiedmicrosatellites is 100%.
 46. The method of claim 42 wherein saidamplification comprises contacting said nucleobase sequence with anenzyme having a polymerase activity.
 47. The method of claim 46 whereinthe enzyme having polymerase activity is selected from the groupconsisting of AmpliTaq Gold® DNA polymerase; AmpliTaq® DNA Polymerase;AmpliTaq® DNA Polymerase, Stoffel fragment; rTth DNA Polymerase; rTthDNA Polymerase XL; Tne, Bst DNA polymerase large fragment from Bacillusstearothermophilus; Vent and Vent Exo- from Thermococcus litoralis; Tmafrom Thermotoga maritima; Deep Vent and Deep Vent Exo- and Pfi fromPyrococcus; and mutants, variants and derivatives thereof.
 48. Themethod of claim 42 wherein said amplified microsatellite is amononucleotide microsatellite.
 49. The method of claim 42 wherein saidamplified microsatellite is a dinucleotide microsatellite.
 50. Themethod of claim 42 wherein said amplified microsatellite is atrinucleotide microsatellite.
 51. The method of claim 42 wherein saidamplified microsatellite is a tetranucleotide microsatellite.
 52. Themethod of claim 42 wherein said amplified microsatellite is apentanucleotide microsatellite.
 53. The method of claim 42 wherein saidincubation is performed in the presence of a set of dNTPs, said setcomprising an amount of dNTP complementary to adenosine, an amount ofdNTP complementary to guanosine, an amount of dNTP complementary tocytidine and an amount of dNTP complementary to thymine, wherein each ofsaid amounts of dNTP is least 0.5 mM.
 54. The method of claim 42 whereinsaid incubation is performed in the presence of a set of dNTPs, said setcomprising an amount of dNTP complementary to adenosine, an amount ofdNTP complementary to guanosine, an amount of dNTP complementary tocytidine and an amount of dNTP complementary to thymine, wherein each ofsaid amounts of dNTP is least 1 mM.
 55. The method of claim 42 whereinsaid sorbitol is present in an amount of 1.5 to 3.5 M.
 56. The method ofclaim 42 wherein said sorbitol is present in an amount of 2.0 to 3.0 M.57. A method comprising the steps of: (a) providing a sample comprisinga nucleic acid that contains one or more microsatellites selected fromthe group consisting of mononucleotide microsatellites, dinucleotidemicrosatellites, trinucleotide microsatellites, tetranucleotidemicrosatellites, and pentanucleotide microsatellites; and (b) amplifyingat least one nucleobase sequence of said nucleic acid, said nucleobasesequence comprising at least one of said microsatellites; said amplifiedmicrosatellite having a G+C content of greater than 50%; wherein saidamplification is performed in the presence of sorbitol in an amount of1.5 to 3.5 M, and in the presence of a set of dNTPs, said set comprisingan amount of dNTP complementary to adenosine, an amount of dNTPcomplementary to guanosine, an amount of dNTP complementary to cytidineand an amount of dNTP complementary to thymine, wherein each of saidamounts of dNTP is least 0.5 mM.
 58. A method for performing polymerasechain reaction amplification of a microsatellite selected from the groupconsisting of mononucleotide microsatellites, dinucleotidemicrosatellites, trinucleotide microsatellites, tetranucleotidemicrosatellites and pentanucleotide microsatellites, said amplifiedmicrosatellite having a G+C content of greater than 50%, said methodcomprising the step of contacting said microsatellite with a polymerasein the presence of an amount of sorbitol effective to reduce the amountof stutter arising from said amplification, relative to the amount ofsuch stutter observed in the absence of sorbitol.
 59. The method ofclaim 58 wherein said polymerase is selected from the group consistingof AmpliTaq Gold® DNA polymerase; AmpliTaq® DNA Polymerase; AmpliTaq®DNA Polymerase, Stoffel fragment; rTth DNA Polymerase; rTth DNAPolymerase XL; Tne, Bst DNA polymerase large fragment from Bacillusstearothermophilus; Vent and Vent Exo- from Thermococcus litoralis; Tmafrom Thermotoga maritima; Deep Vent and Deep Vent Exo- and Pfu fromPyrococcus; and mutants, variants and derivatives thereof.
 60. Themethod of claim 58 wherein said incubation is performed in the presenceof a set of dNTPs, said set comprising an amount of dNTP complementaryto adenosine, an amount of dNTP complementary to guanosine, an amount ofdNTP complementary to cytidine and an amount of dNTP complementary tothymine, wherein each of said amounts of dNTP is least 0.5 mM.
 61. Themethod of claim 58 wherein said incubation is performed in the presenceof a set of dNTPs, said set comprising an amount of dNTP complementaryto adenosine, an amount of dNTP complementary to guanosine, an amount ofdNTP complementary to cytidine and an amount of dNTP complementary tothymine, wherein each of said amounts of dNTP is least 1 mM.
 62. Themethod of claim 58 wherein said sorbitol is present in an amount of 1.5to 3.5 M.
 63. The method of claim 58 wherein said sorbitol is present inan amount of 2.0 to 3.0 M.
 64. A composition comprising: (a) a nucleicacid sequence comprising a microsatellite, said microsatellite having aG+C content of greater than 50%, said microsatellite being selected fromthe group consisting of mononucleotide microsatellites, dinucleotidemicrosatellites, trinucleotide microsatellites, tetranucleotidemicrosatellites, and pentanucleotide microsatellites; (b) at least twoprimers, each of said primers having a sequence that is substantiallycomplementary to a portion of said nucleic acid sequence that isadjacent to said microsatellite; (c) at least one enzyme having nucleicacid polymerase activity; and (d) sorbitol.
 65. The method of claim 64wherein said incubation is performed in the presence of a set of dNTPs,said set comprising an amount of dNTP complementary to adenosine, anamount of dNTP complementary to guanosine, an amount of dNTPcomplementary to cytidine and an amount of dNTP complementary tothymine, wherein each of said amounts of dNTP is least 0.5 mM.
 66. Themethod of claim 64 wherein said incubation is performed in the presenceof a set of dNTPs, said set comprising an amount of dNTP complementaryto adenosine, an amount of dNTP complementary to guanosine, an amount ofdNTP complementary to cytidine and an amount of dNTP complementary tothymine, wherein each of said amounts of dNTP is least 1 mM.
 67. Themethod of claim 64 wherein said sorbitol is present in an amount of 1.5to 3.5 M.
 68. The method of claim 64 wherein said sorbitol is present inan amount of 2.0 to 3.0 M.
 69. A kit for the amplification of a targetnucleic acid sequence, said target nucleic acid sequence comprising amicrosatellite selected from the group consisting of mononucleotidemicrosatellites, dinucleotide microsatellites, tetranucleotidemicrosatellites, and pentanucleotide microsatellites, saidmicrosatellite having a G+C content of greater than 50%, comprising, inseparate containers: a polymerase, a plurality of deoxynucleotidetriphosphates; and sorbitol.
 70. The kit of claim 69 wherein saidpolymerase is selected from the group consisting of AmpliTaq Gold® DNApolymerase; AmpliTaq® DNA Polymerase; AmpliTaq® DNA Polymerase, Stoffelfragment; rTth DNA Polymerase; rTth DNA Polymerase XL; Tne, Bst DNApolymerase large fragment from Bacillus stearothermophilus; Vent andVent Exo- from Thermococcus litoralis; Tma from Thermotoga maritima;Deep Vent and Deep Vent Exo- and Pfu from Pyrococcus; and mutants,variants and derivatives thereof.
 71. A method of detecting cancer, apre-cancerous condition or genetic disorder in a subject comprisingamplifying a region of DNA from a subject, wherein said region comprisesa microsatellite selected from the group consisting of a mononucleotiderepeat, a dinucleotide repeat, a trinucleotide repeat, a tetranucleotiderepeat, and a pentanucleotide repeat, wherein said amplificationcomprises the steps of: (a) providing a sample comprising a nucleic acidthat contains a nucleic acid having a microsatellite instability, (b)amplifying at least one nucleobase sequence of said nucleic acid, saidnucleobase sequence comprising at least one of said microsatellites; and(c) detecting alterations of said microsatellite as compared tocorresponding microsatellites amplified from control tissue; saidamplified microsatellite having a G+C content of greater than 50%;wherein said amplification is performed in the presence of a sufficientamount of sorbitol, effective to reduce said stutter relative to theamount of stutter observed in the absence of sorbitol.
 72. The method ofclaim 71 wherein said cancer or cancerous condition is selected from thegroup consisting of chronic lymphocytic leukemia.
 73. The method ofclaim 71 wherein said genetic disorder is selected from the groupconsisting of oculopharyngeal muscular dystrophy, myotonic dystrophy,and Fragile X Syndrome.
 74. The method of claim 72 wherein said regioncomprises a genetic locus comprising a repeat selected from the groupconsisting of CAG/CTG, CCG/CGG, and CGA/TCG.
 75. The method of claim 73wherein said region comprises a genetic locus comprising a repeatselected from the group consisting of CAG/CTG, and GCG/CGC.
 76. Themethod of claim 71 wherein said incubation is performed in the presenceof a set of dNTPs, said set comprising an amount of dNTP complementaryto adenosine, an amount of dNTP complementary to guanosine, an amount ofdNTP complementary to cytidine and an amount of dNTP complementary tothymine; wherein each of said amounts of dNTP is least 0.5 mM.
 77. Themethod of claim 71 wherein said incubation is performed in the presenceof a set of dNTPs, said set comprising an amount of dNTP complementaryto adenosine, an amount of dNTP complementary to guanosine, an amount ofdNTP complementary to cytidine and an amount of dNTP complementary tothymine; wherein each of said amounts of dNTP is least 1 mM.
 78. Themethod of claim 71 wherein said sorbitol is present in an amount of 1.5to 3.5 M.
 79. The method of claim 71 wherein said sorbitol is present inan amount of 2.0 to 3.0 M.
 80. A method of gene typing comprisingamplifying a plurality of regions of DNA from a sample containing DNAfrom a subject, wherein said regions comprise at least onemicrosatellite selected from the group consisting of a mononucleotiderepeat, a dinucleotide repeat, a trinucleotide repeat, a tetranucleotiderepeat, and a pentanucleotide repeat, said microsatellite having a G+Ccontent of greater than 50%, wherein said amplification comprises thesteps of: (a) contacting said DNA with a enzyme at least one enzymehaving nucleic acid polymerase activity; (b) incubating said sample withsaid enzyme for a time and under conditions sufficient to amplify saidregions; and (c) separating amplified regions, forming a microsatellitepattern; wherein said incubation is performed in the presence of anamount of sorbitol effective to reduce said stutter relative to theamount of stutter observed in the absence of sorbitol.
 81. The method ofclaim 80 wherein said incubation is performed in the presence of a setof dNTPs, said set comprising an amount of dNTP complementary toadenosine, an amount of dNTP complementary to guanosine, an amount ofdNTP complementary to cytidine and an amount of dNTP complementary tothymine; wherein each of said amounts of dNTP is least 0.5 mM.
 82. Themethod of claim 80 wherein said incubation is performed in the presenceof a set of dNTPs, said set comprising an amount of dNTP complementaryto adenosine, an amount of dNTP complementary to guanosine, an amount ofdNTP complementary to cytidine and an amount of dNTP complementary tothymine; wherein each of said amounts of dNTP is least 1 mM.
 83. Themethod of claim 80 wherein said sorbitol is present in an amount of 1.5to 3.5 M.
 84. The method of claim 80 wherein said sorbitol is present inan amount of 2.0 to 3.0 M.
 85. A method of personal geneticidentification comprising amplifying a plurality of regions of DNA froma sample containing DNA from a subject, wherein said regions comprise atleast one microsatellite selected from the group consisting of amononucleotide repeat, a dinucleotide repeat, a trinucleotide repeat, atetranucleotide repeat, and a pentanucleotide repeat, wherein saidmicrosatellite has a G+C content of greater than 50%, wherein saidamplification comprises the steps of: (a) contacting said DNA with aenzyme at least one enzyme having nucleic acid polymerase activity; and(b) incubating said sample with said enzyme for a time and underconditions sufficient to amplify said regions; (c) separating amplifiedregions, forming a microsatellite pattern; and (d) comparing saidmicrosatellite pattern with a corresponding microsatellite patternderived from the a DNA sample from a second source; wherein saidincubation is performed in the presence of an amount of an sorbitoleffective to reduce said stutter relative to the amount of stutterobserved in the absence of sorbitol.
 86. The method of claim 85 whereinsaid incubation is performed in the presence of a set of dNTPs, said setcomprising an amount of dNTP complementary to adenosine, an amount ofdNTP complementary to guanosine, an amount of dNTP complementary tocytidine and an amount of dNTP complementary to thymine; wherein each ofsaid amounts of dNTP is least 0.5 mM.
 87. The method of claim 85 whereinsaid incubation is performed in the presence of a set of dNTPs, said setcomprising an amount of dNTP complementary to adenosine, an amount ofdNTP complementary to guanosine, an amount of dNTP complementary tocytidine and an amount of dNTP complementary to thymine; wherein each ofsaid amounts of dNTP is least 1 mM.
 88. The method of claim 85 whereinsaid sorbitol is present in an amount of 1.5 to 3.5 M.
 89. The method ofclaim 85 wherein said sorbitol is present in an amount of 2.0 to 3.0 M.